Methods for imaging biological samples, such as tissue sections, are important for many medical applications, including diagnostics, disease monitoring, prognosis, and drug discovery. With the current growth and future potential of personalized medicine, there is an increasing demand for rapid, high-throughput and sensitive methods to detect a large number of disease- and individual-specific biomarkers in order to provide personalized diagnoses and therapies to patients. However, current imaging methods are limited in their multiplexing capabilities, speed, resolution and sensitivity, and by high cost.
Fluorescence microscopy is a well-known method for imaging cells and detecting biomarkers based on optical properties of fluorescently labeled samples. However, fluorescence microscopy is limited in the number of fluorescent labels that can be used simultaneously because of the spectral overlap between different labels, and is limited in resolution by the diffraction limit of light (at about 0.2 μm).
As an alternative to detecting optical signals from a sample, methods to detect molecular mass signatures of a sample using mass spectrometry are known. For example, in matrix assisted laser desorption ionization (MALDI) mass spectrometry, a sample is embedded in an appropriate matrix and irradiation of the sample with a laser beam causes desorption and ionization of molecules in the sample due to absorption of photon energy by the matrix. The released ions are extracted from the source and detected in a mass spectrometer. However, MALDI has low ionization efficiency on the order of 10−6 to 10−3, which limits sensitivity, as well as a complex process for sample preparation, and therefore is not amenable to high-throughput analysis.
Another mass spectrometry imaging method is secondary ion mass spectrometry (SIMS), in which a primary ion beam is applied to the sample to sputter secondary ions, which can be detected using a mass spectrometer. However, the efficiency of ionization depends on the primary ion species, and is on average only 1% of the total sputtered species, which include secondary ions and neutral species. Such low ionization efficiency limits the speed with which a sample may be imaged at a given sensitivity. On the other hand, primary ions that are more efficient at ionization, such as oxygen, require bulky, expensive setups to generate the ion beam.
In addition, the number of endogenous targets that can be detected simultaneously by mass spectrometry imaging techniques is limited by the ability to resolve mass signatures of the ionized species.
Thus there is a need for improved, cost effective methods for highly multiplexed, high-throughput and high-resolution imaging of biological samples.